NAP (davunetide) rescues neuronal dysfunction in a Drosophila model of tauopathy.

Alzheimer's disease (AD) is a devastating neurodegenerative disease causing irreversible cognitive decline in the elderly. There is no disease-modifying therapy for this condition and the mechanisms underpinning neuronal dysfunction and neurodegeneration are unclear. Compromised cytoskeletal integrity within neurons is reported in AD. This is believed to result from loss-of-function of the microtubule-associated protein tau, which becomes hyper-phosphorylated and deposits into neurofibrillary tangles in AD. We have developed a Drosophila model of tauopathy in which abnormal human tau mediates neuronal dysfunction characterised by microtubule destabilisation, axonal transport disruption, synaptic defects and behavioural impairments. Here we show that a microtubule-stabilising drug, NAPVSIPQ (NAP), prevents as well as reverses these phenotypes even after they have become established. Moreover, it does not alter abnormal tau levels indicating that it by-passes toxic tau altogether. Thus, microtubule stabilisation is a disease-modifying therapeutic strategy protecting against tau-mediated neuronal dysfunction, which holds great promise for tauopathies like AD.

Increased throughput assays of locomotor dysfunction in Drosophila larvae.

Larval locomotion is a sensitive readout of a range of nervous system deficits in Drosophila, and has been utilised to quantify modulation of the disease phenotype in models of human disease. Single larvae are typically analysed in series using manual quantification of parameters such as contraction rate, or grouped together and studied en-masse. Here, we describe the development of tests for the analysis of several spatially isolated third instar larvae in parallel. We rapidly quantify larval turning rate and velocity during wandering behaviour in a 4 plate assay. In a second test, larvae are recorded as they race along five parallel lanes towards a yeast stimulus. This allows increased throughput analysis of comparative genotypes simultaneously, video archiving, and detection of exacerbation or rescue of defective locomotion in a Drosophila model of tauopathy, as we demonstrate genetically and through delivery of candidate therapeutic chemicals in fly food. The tests are well-suited for rapid comparison of locomotion capability in Drosophila mutants or candidate modulation screens in Drosophila models of human disease.

Relationship between individual herd-heritability estimates and sire misidentification rate.

The objectives of this study were to estimate heritabilities within herds participating in Dairy Herd Improvement and determine the relationship of the individual herd heritability with sire misidentification rate. Individual herd heritabilities for milk, fat, and protein yield and somatic cell score (SCS) were calculated with daughter-dam regression and daughter-sire predicted transmitting ability (PTA) regression using 4,712,166 records from 16,336 herds available for August 2000 evaluations and 7,084,953 records from 20,920 herds available for August 2006 evaluations. Herd heritabilities were estimated using regression models that included fixed breed, age within parity, herd-year-season of calving, dam records nested within state, sire PTA within state, and an interaction between sire PTA and herd variance; random regression coefficients were dam records within herd and sire PTA within herd. Average daughter-dam herd heritability estimates ranged from 0.21 (SCS in 2000) to 0.73 (protein percentage in 2006), whereas daughter-sire herd heritability ranged from 0.10 (SCS in 2000) to 0.42 (protein percentage in 2006). Verification of sire identification with DNA marker analysis was provided by Accelerated Genetics and Alta Genetics Inc. Daughter-sire herd heritability was more strongly correlated with sire misidentification rate than daughter-dam herd heritability. The correlation between the first principal component for all measures of herd heritability and sire misidentification rate was -0.38 (176 herds) and -0.50 (230 herds) in 2000 and 2006, respectively. Herd heritability can be estimated with simple regression techniques for several thousand herds simultaneously. The herd heritability estimates were correlated negatively with sire misidentification rates and could be used to identify herds that provide inaccurate data for progeny testing.

A prospective, randomised, single-blind pilot study to determine the effect of anaesthetic technique on troponin T release after off-pump coronary artery surgery.

Ischaemic damage to the myocardium inevitably occurs during coronary artery surgery. However, the extent of the damage may be influenced by the anaesthetic technique used. The most sensitive and reliable marker of myocardial damage is currently thought to be troponin T. We conducted a prospective, randomised, single-blind pilot study to determine the baseline values of troponin T release after off-pump coronary artery bypass surgery in 30 patients randomly allocated to receive either propofol, isoflurane or isoflurane and high thoracic epidural analgesia. All other treatment was standardised. Patients undergoing emergency surgery and those with unstable angina were excluded. Blood samples were taken at 0, 3, 6, 12, 24 and 48 h after surgery for troponin T analysis. Mean troponin T levels at 24 h were not significantly different between the groups (p = 0.41). These data allows appropriate power calculations for further, large-scale studies to determine the anaesthetic technique that provides optimal myocardial protection.

TYRP1 is associated with dun coat colour in Dexter cattle or how now brown cow?

Tyrosinase related protein 1 (TYRP1), which is involved in the coat colour pathway, was mapped to BTA8 between microsatellites BL1080 and BM4006, using a microsatellite in intron 5 of TYRP1. The complete coding sequence of bovine TYRP1 was determined from cDNA derived from skin biopsies of cattle with various colours. Sequence data from exons 2-8 from cattle with diluted phenotypes was compared with that from non-diluted phenotypes. In addition, full-sib families of beef cattle generated by embryo transfer and half-sib families from traditional matings in which coat colour was segregating were used to correlate TYRP1 sequence variants with dilute coat colours. Two non-conservative amino acid changes were detected in Simmental, Charolais and Galloway cattle but these polymorphisms were not associated with diluted shades of black or red, nor with the dun coat colour of Galloway cattle or the taupe brown colour of Braunvieh and Brown Swiss cattle. However, in Dexter cattle all 25 cattle with a dun brown coat colour were homozygous for a H424Y change. One Dexter that was also homozygous Y434 was red because of an "E+/E+" genotype at MC1R which lead to the production of only phaeomelanin. None of the 70 remaining black or red Dexter cattle were homozygous for Y434. This tyrosine mutation was not found in any of the 121 cattle of other breeds that were examined.

Comparison of intrathecal fentanyl and diamorphine in addition to bupivacaine for caesarean section under spinal anaesthesia.

BACKGROUND: Co-administration of small doses of opioids and bupivacaine for spinal anaesthesia reduces intraoperative discomfort and may reduce postoperative analgesic requirements in patients undergoing Caesarean section. Fentanyl and diamorphine are the two most frequently used agents in UK obstetric anaesthetic practice. METHODS: Seventy-five healthy parturients scheduled for elective Caesarean section under spinal anaesthesia using hyperbaric 0.5% bupivacaine, were randomly allocated to additionally receive intrathecal fentanyl 20 micrograms, diamorphine 300 micrograms or 0.9% saline. Patients also received i.v. cyclizine and rectal diclofenac. RESULTS: Less supplementary intraoperative analgesia was required by patients in either opioid group (4%) compared with the control (32%) (P < 0.05). Twenty four hours after spinal injection, total mean (SD) postoperative morphine requirement was significantly lower if diamorphine was administered (31 (21) mg), in comparison with the other two groups (control 68 (26) mg; fentanyl 62 (26) mg) (P < 0.05). Reduced visual analogue pain scores were evident 12 h following diamorphine, but observed only for 1 h after fentanyl when compared with the control (P < 0.05). Mild pruritus was more common for 2 h after either spinal opioid (P < 0.05), but no inter-group differences were observed for the remainder of the first 24 h. Patients displayed deeper levels of sedation both acutely and 12 h after administration of intrathecal fentanyl (P < 0.05). CONCLUSIONS: Both intrathecal opioids reduce intraoperative discomfort, but only diamorphine reduced postoperative analgesic requirement beyond the immediate postoperative period.

Sedation and analgesia for transvaginal oocyte retrieval: an audit resulting in a change of clinical practice.

The aim was to audit the standard of care at oocyte retrieval with respect to the analgesia used. Before June 1999, a combination of intramuscular pethidine and prochlorperazine was given as a single dose. Many women found this to be unsatisfactory and requested general anaesthesia for subsequent oocyte retrievals. An intravenous sedation and analgesia regimen was introduced using a combination of midazolam and fentanyl. The introduction of this regimen was audited and compared with the established regimen. This study describes the experience with this technique, its patient acceptability, safety and cost implications.

Caspases 3 and 9 send a pro-apoptotic signal from synapse to cell body in olfactory receptor neurons.

Caspase-9, an initiator caspase, and caspase-3, an effector caspase, have been suggested to mediate the terminal stages of neuronal apoptosis, but little is known about their activation in vivo. We examined temporal and spatial aspects of caspase-9 and -3 activation in olfactory receptor neurons (ORNs) undergoing apoptosis after target removal in vivo. After removal of the olfactory bulb, enhanced expression of procaspase-9 and -3 is observed in ORNs, followed by activation initially at the level of the lesion, then in axons, and only later in the ORN soma. We established the amyloid precursor-like protein-2 (APLP2) as a caspase substrate that is cleaved in an identical spatiotemporal pattern, suggesting its cleavage is the result of retrograde propagation of a pro-apoptotic signal in a caudorostral wave from the synapse through the axon to the ORN cell body. A null mutation in caspase-3 causes a change in axonal patterning indicative of an overall developmental expansion of the ORN population, and mature ORNs of caspase-3 knock-outs do not undergo caspase-dependent terminal dUTP nick end labeling-positive apoptosis after olfactory bulb removal. These results demonstrate that ORNs require caspase-3 activation to undergo normal developmental and mature target-deprived apoptosis. In addition, we demonstrate an axonal site of action for caspase-3 and -9 and show that regulation and activation of caspase-3 and -9 leading to apoptosis is a highly ordered process that occurs initially at the presynaptic level and only later at the cell body after deafferentation.

Prevention and management of hypotension during spinal anaesthesia for elective Caesarean section: a survey of practice.

Hypotension during obstetric spinal anaesthesia has traditionally been managed by such measures as fluid preloading, positioning of the patient and the use of vasoconstrictors. However, studies and reports have regularly appeared in the literature disputing the value of conventional management, in particular, the fluid preload. With this in mind, we surveyed UK consultant obstetric anaesthetists to determine current practice in this area. Of the 558 respondents, 486 (87.1%) stated that they routinely give a fluid preload. The fluid chosen by 405 (83.3%) of the preloaders was Hartmann's solution and the usual volume, chosen by 194 (39.9%), was 1000 ml. A simple left lateral position was preferred by 221 respondents (39.6%) overall and in the treatment of hypotension, ephedrine was the sole vasoconstrictor selected by 531 (95.2%). Heavy bupivacaine 0.5% was the local anaesthetic chosen by 545 (97.7%) and 407 (72.9%) respondents indicated the use of additional spinal drugs.

A survey of epidural technique and accidental dural puncture rates among obstetric anaesthetists.

Five hundred UK obstetric anaesthetists were surveyed to investigate retrospectively the relationship between experience, rotation of the epidural needle within the epidural space, choice of loss-of-resistance agent and accidental dural puncture (ADP) rate. Responses were received from 390 (78%) of the members surveyed. Anaesthetists with more than 15 years' experience are more likely to perform an epidural with the patient in the lateral position (P < 0.001), use loss-of-resistance to air to detect the epidural space (P < 0.001) and rotate the epidural needle after identifying the epidural space (P = 0.001) when compared to those of less experience. A reduced inadvertent dural puncture rate was found to be associated with increased frequency of performing the procedure (P = 0.012), greater experience of the practitioner (P = 0.049) and non-rotation of the epidural needle (P = 0.023). There are three components that can alter from case to case; patient positioning, loss-of-resistance agent and needle rotation. Loss-of-resistance agent and patient positioning in isolation did not significantly influence ADP rate. This study suggests that the combination of practising lateral patient positioning, loss-of-resistance to saline and non-rotation of the epidural needle significantly reduces ADP rate (P = 0.035).

Spinal anaesthesia for caesarean section: current clinical practice.

Spinal anaesthesia has evolved as the preferred anaesthetic technique for most cases of caesarean section. Having been extensively studied and refined over the years, there are few situations where a spinal is absolutely contraindicated. While general anaesthesia will always have a place in obstetrics, in experienced hands a spinal offers safety, efficacy and an improvement in maternal morbidity.

Cancer risk in women exposed to diethylstilbestrol in utero.

CONTEXT: The association between in utero exposure to diethylstilbestrol (DES) and clear cell adenocarcinoma (CCA) of the vagina and cervix is well known, yet there has been no systematic study of DES-exposed daughters to determine whether they have an increased risk of other cancers. As many as 3 million women in the United States may have been exposed to DES in utero. OBJECTIVE: To determine whether women exposed to DES in utero have a higher risk of cancer after an average of 16 years of follow-up. DESIGN: A cohort study with mailed questionnaires and medical record review of reported cancer outcomes. PARTICIPANTS: A cohort of 4536 DES-exposed daughters (of whom 81% responded) and 1544 unexposed daughters (of whom 79% responded) who were first identified in the mid-1970s. MAIN OUTCOME MEASURES: Cancer incidence in DES-exposed daughters compared with population-based rates and compared with cancer incidence in unexposed daughters. RESULTS: To date, DES-exposed daughters have not experienced an increased risk for all cancers (rate ratio, 0.96; 95% confidence interval [CI], 0.58-1.56) or for individual cancer sites, except for CCA. Three cases of vaginal CCA occurred among the exposed daughters, resulting in a standardized incidence ratio of 40.7 (95% CI, 13.1-126.2) in comparison with population-based incidence rates. The rate ratio for breast cancer was 1.18 (95% CI, 0.56-2.49); adjustment for known risk factors did not alter this result. CONCLUSIONS: Thus far, DES-exposed daughters show no increased cancer risk, except for CCA. Nevertheless, because exposed daughters included in our study were, on average, only 38 years old at last follow-up, continued surveillance is warranted to determine whether any increases in cancer risk occur during the menopausal years.

Relationship of bovine leukocyte adhesion deficiency with genetic merit for performance traits.

Examination of the existence of pleiotropy or linkage of bovine leukocyte adhesion deficiency with other traits and of the impact of removal of the recessive, undesirable allele on genetic progress for those traits has been limited. Frequency of carriers among 6400 Holstein bulls tested was 8.2%; however, reporting was incomplete, and, therefore, the estimate of carrier frequency was biased downward. For AI-sampled bulls, carrier frequency reached a high of 23% for bulls sampled during 1989 but declined to 0% since then because of DNA testing and culling. Association of the allele with yield, productive life, and somatic cell score was examined with a model in which the daughter yield deviation minus the mean of parent evaluations was explained by carrier status. A significant negative relationship was found with protein yield when effect of sires was ignored; all associations were unfavorable. Linkage was examined by applying the model for each of four sire families; only protein yield for one sire was significantly and negatively related to the recessive allele. Carrier bulls currently are labeled, and some continue to be used actively in AI because of superiority for other traits. Consequential pleiotropy of the allele or linkage of the locus with the traits studied is unlikely. Genetic progress for these performance traits will not be impeded by failure to sample carrier bulls.

Chromosome substitution effects associated with kappa-casein and beta-lactoglobulin in Holstein cattle.

Allelic variants of kappa-casein and beta-lactoglobulin were used to estimate chromosome substitution effects on transmitting abilities for yield traits among sons of two Holstein sires. Sire 1 was a heterozygote for both loci, whereas sire 2 was a heterozygote for beta-lactoglobulin only. Transmitting abilities for fat percentage and the exchange of protein and fat (protein yield minus fat yield) were influenced by kappa-casein genotype among offspring of sire 1. The B allele of kappa-casein was associated with a decrease in fat percentage, whereas fat yield was not affected; the result was 4.5 kg more transmitting ability for protein than for fat. Favorable chromosome substitution effects on transmitting abilities for fat percentage, protein yield, and the exchange of protein and fat were also observed for the A allele of beta-lactoglobulin among offspring of sire 1; there were consistent but not significant estimates for sire 2. These results suggest that different estimates could be obtained from offspring of different parents, perhaps from linkage disequilibrium between families. The chromosome substitution effects appeared to be in the direction opposite to the average correlation of milk fat and protein content; marker-assisted selection for increased yield of protein would result in decreased fat yield.

Validation and application of an assay for deoxyribonucleic acid to estimate concentrations of bull sperm.

Spectrophotometers are used for estimating sperm concentrations from raw ejaculates in semen processing laboratories. Unfortunately, these instruments have a limited detection spectrum and do not permit accurate quantification of sperm numbers in highly diluted or concentrated samples. The objectives of this study were to validate a DNA assay for quantification of sperm numbers in extended or undiluted semen samples and to determine precision of the assay. The principle of the assay is based upon a fluorescent dye that binds to adenine-thymine base pairs in double-stranded DNA. Semen samples and calf thymus DNA standards were sonicated in 2 M NaCl buffer with 1 mM EDTA. The DNA content of samples was compared to standards of calf thymus DNA using fluorometry. Sensitivity of the assay was determined to be 1.4 x 10(5) sperm cells. Concentrations of sperm estimated from DNA assay values did not differ from flow cytometric cell counts. Assays were performed in three different laboratories, using different equipment, to assess the assay's repeatability. Estimates of sperm concentrations determined by the DNA assay were similar, regardless of location and source of equipment used to perform the assays. This assay fulfills statistical criteria for being sensitive, accurate, and repeatable, and it can be employed in laboratories processing semen for artificial insemination as a tool for spectrophotometer calibration, a check for straw filling accuracy, or to quantify sperm numbers in extended, packaged semen.

Structural variation around prolactin gene linked to quantitative traits in an elite Holstein sire family.

Digestion of genomic DNA with the restriction endonuclease Avail disclosed a probable insertion deletion of approximately 200 base pairs (bp) near the prolactin gene. Two alleles were apparent as three distinct hybridization patterns. These alleles were statistically associated with quantitative trait loci among sons of one elite Holstein sire family. The favorable genotype was correlated with the presence of a 1.15-kb hybridization band inherited from the sire when genomic DNA was probed with a full-length cDNA for prolactin. Pedigree estimates of genetic merit among genotypes were similar, differing by only 19.3 kg for milk in ancestor merit. Comparisons of genetic estimates for quantitative yield traits in offspring of this heterozygous sire showed significant (P<0.05) differences between homozygous genotypes for predicted difference milk (PDM), predicted difference dollars (PD$), cheese yield dollars, and protein dollars. The estimated differences between homozygous genotypes for USDA Transmitting Abilities of PDM, PD$, Cheese Yield $ and Protein $ were 282.93 kg, $74.35, $48.58 and $53.67, respectively. However, the estimated breeding values from progeny ranged over 900 kg in transmitting ability for milk. Frequency of the favorable marker allele was estimated to be 0.231 in the elite cow population used as dams of sons. These results demonstrate the potential of molecular biological techniques to discriminate between individuals within a family and to predict breeding values for selection schemes.

An analytical model for the estimation of chromosome substitution effects in the offspring of individuals heterozygous at a segregating marker locus.

Use of marker genes for quantitative traits has been suggested as a supplement to selection for livestock species. Linkage relationships can be estimated by using data from offspring of a heterozygous parent, if offspring can be positively assigned segregation of one or the other of the marker alleles. In field data, some data on offspring can be characterized and used to estimate the difference in chromosome substitution effects, but other matings result in uncertain transfer of the marker alleles. In this study, an alternative estimation procedure is proposed that would allow incorporation of data on all offspring of a heterozygous parent, even those where chromosome segregation is ambiguous. If the frequency of the marker alleles is known in the population of mates of a heterozygous individual, the mean and variance of the heterozygous offspring can be used in a generalized leastsquares model to estimate the chromosome substitution effect. When gene frequencies are not known, maximum likelihood estimates can be obtained from the data for use in a conditional estimate. Monte Carlo simulations of data following the assumed genetic model were analyzed as proposed, and parameter estimates were characterized. Estimates of chromosome substitution effects were reasonable approximations of input values. Distributions of t-statistics testing the null hypothesis of no difference between marked chromosome segments were unbiased, with only slightly larger variance than expected. Addition of data from heterozygous offspring improved the efficiency of detection of chromosome substitution effects by more than four times when marker gene frequencies were low.

Restriction fragment length polymorphisms associated with growth hormone and prolactin genes in Holstein bulls: evidence for a novel growth hormone allele.

Sperm DNA isolated from sons of three extensively used US Holstein bulls was screened for differences associated with the primary gene structure of the bovine growth hormone (bGH) and prolactin (bPrl) genes. Southern blot analysis of DNA digested with 10 restriction enzymes revealed that offspring from two of the three bull families exhibited polymorphisms around the bGH and bPrl genes. Restriction fragment length polymorphisms (RFLPs) around the bGH gene were detected with five enzymes, whereas three enzymes revealed RFLPs around the bPrl gene. At least three structural differences were predicted around the bGH gene. The most common variant hybridization pattern appeared to involve an insertion/deletion located downstream of the conserved 3' EcoRI site. The presence of RFLPs in the genes coding for these pituitary hormones within a familial line may provide the basis for genetic markers associated with lactation and mammary development.